Midkine (MK) is a heparin binding growth factor of about 13 kDa rich in basic amino acids and cysteine which was discovered in 1988 by Kadomatsu et al. as a product of a gene expressed temporarily during the differentiation induction process of embryonal tumor cells by retinoic acid (NPLs 1 and 2).
Midkine is widely present in vertebrates, and is reported in humans, rats, mice, rabbits, cattle, fowls, xenopus and zebra fish. Its conservation is high and, for example, human midkine and mouse midkine have an amino acid homology of 87% in the entire molecule (NPL 3).
Activities such as heparin-binding capacity, neurite outgrowth, and neuronal migration are borne by a half molecule on the C-terminal side including a C-domain (NPL 4). The C-domain of human midkine and that of mouse midkine have an amino acid homology of 93% (40/43).
Midkine forms an independent family composed of pleiotrophin (PTN) as the only constituent. Pleiotrophin is a polypeptide of about 18 kDa, has an amino acid homology of about 45% to midkine, and promotes the elongation and growth of neurites, promotes transformation of cells, and promotes neovascularization.
Midkine is characterized by being expressed on the epithelium side of tissue where epithelial-stromal interaction occurs in the embryonal period (NPL 5). Its expression peaks in the middle of the embryonal period, then gradually decreases, is low at birth, and minimizes in an adult. However, strong expression is induced during the process of carcinogenesis, inflammation, or repair.
Midkine is known to have various biological functions. Broadly, midkine is important in three fields, cancer, inflammation, and nerve.
Midkine is considered to promote the survival and migration of cancer cells, trigger neovascularization, and assist in the progression of cancer. The manner of midkine expression in human cancer is characterized in that its expression is accelerated at an incidence exceeding 70%, regardless of the type of cancer. This is confirmed in a variety of cancers including esophageal carcinoma, thyroid carcinoma, bladder cancer, stomach cancer, pancreatic cancer, liver cancer, pulmonary cancer, breast cancer, neuroblastoma, glioblastoma, uterine cancer, ovarian cancer, and Wilms tumor (for example, NPLs 6 to 10).
Neuroblastoma is one of neuroendocrine tumors. It is extracranial solid cancer with the highest incidence in childhood. It is the fourth most frequent childhood malignant tumor following leukemia, central nervous system tumor, and lymphoma. Neuroblastoma is one of the three major causes of cancer deaths in children, and is the greatest cause of cancer deaths in infants. In the United States, about 650 new cases of neuroblastoma occur in a year. Approximately 50% of the neuroblastoma cases occur in children of less than 2 years of age. As a characteristic of neuroblastoma with a poor prognosis, N-myc gene is known to proliferate (NPLs 11 to 13).
There is a correlation between a neuroblastoma prognosticator, such as N-myc gene proliferation, and the blood midkine level (NPL 14). Furthermore, blood midkine can serve, alone, as a prognostic factor for neuroblastoma (NPL 15).
Midkine has the function of promoting inflammation. This is based mainly on findings obtained from the analysis of midkine knockout mice (Mdk−/−). In the knockout mouse, for example, the formation of a neointima at the time of injury to the blood vessel and the occurrence of nephritis associated with ischemic injury are alleviated. The condition of rheumatism models or postoperative adhesion is also relieved greatly. Moreover, midkine is known to promote the migration of inflammatory cells (chemotaxis), such as macrophages and neutrophils, or cause the differentiation of osteoclasts. Based on such findings, midkine is presumed to be involved in inflammatory diseases such as arthritis, autoimmune disease, articular rheumatism (rheumatoid arthritis (RA), osteoarthritis (OA)), multiple sclerosis, postoperative adhesion, inflammatory colitis, psoriasis, lupus, asthma, and neutrophil dysfunction (PTL 1, 2, 3).
Since midkine has intimal thickening action, moreover, it partakes in vascular obstructive diseases such as restenosis after revascularization, coronary vascular obstructive disease, cerebrovascular obstructive disease, renovascular obstructive disease, peripheral vessel obstructive disease, arteriosclerosis, and cerebral infarction (PTL 1).
An example of the function of midkine in the nerve is to assist in the survival of neurocytes and promote the outgrowth of neurites (NPLs 16, 17). For example, when coated with midkine in a lattice form on a culture dish, nerve cells survive in a lattice pattern along the midkine, and extend neurites. Also, midkine is temporarily induced around a lesion in the event of cerebral ischemia (NPL 18), and it is one of cytokines whose expression is induced most after injury in a rat spinal injury model. Midkine induced in this manner is assumed to prevent neuronal death.
The tissue protecting action of midkine during infarction is the same in the heart. On the occasion of acute myocardial infarction, midkine is expressed and induced around the infarct. In a midkine knockout mouse, enlargement of the infarct is observed compared with a wild type mouse, and when in infarction, direct injection of midkine protein into the myocardium preferentially shrinks the infarct and improves cardiac function (NPL 19). Such a protective effect is presumed to owe much to the anti-apoptotic activity of midkine.
Midkine is composed of an N-terminal side fragment (hereinafter, “N-fragment”) comprising amino acids at the 1- to 52-positions, a C-terminal side fragment (hereinafter, “C-fragment”) comprising amino acids at the 62- to 121-positions, and a loop region binding them together (amino acids at the 53- to 61-positions), and its stereostructure is analyzed by NMR (NPL 16). The N-fragment and the C-fragment are constituted, respectively, by a portion having a stereostructure comprising mainly three inverse β-sheets (hereinafter, “domain”), and a portion located outside the domain and having no particular stereostructure (hereinafter, “tail”). The N-fragment is composed of an N-domain comprising amino acids at the 15- to 52-positions and an N-tail comprising amino acids at the 1- to 14-positions, whereas the C-fragment is composed of a C-domain comprising amino acids at the 62- to 104-positions and a C-tail comprising amino acids at the 105- to 121-positions. On the surface of the C-domain, basic amino acids form two clusters. They are a cluster comprising lysine at the 79-position, arginine at the 81-position, and lysine at the 102-position (cluster I), and a cluster comprising lysine at the 86-position, lysine at the 87-position, and arginine at the 89-position (cluster II) (NPL 20). These clusters take part in the heparin-binding capacity of midkine (NPLs 20, 21).
Pharmaceuticals containing midkine inhibitors are disclosed in a plurality of patent gazettes (PTLs 4, 5, 6, 7). However, there are no midkine inhibitors marketed as pharmaceuticals.